GFP-SA融合蛋白的表达纯化及其锚定修饰肿瘤细胞的研究

目的　GFP（绿色荧光蛋白）-SA（链亲和素）双功能融合蛋白的制备及其鉴定研究，以展示我们建立的技术平台，即用含链亲和素的双功能融合蛋白对生物素化的细胞表面进行高效的锚定修饰。方法　构建原核表达载体pET24d/GFP-SA转化大肠杆菌BL21(DE3)。用IPTG诱导重组蛋白的表达，用镍金属螯合（Ni-NTA）层析柱进行纯化。用制备的GFP-SA双功能融合蛋白，对B16肿瘤细胞已生物素化的细胞表面进行修饰，经荧光显微镜和流式细胞仪进行修饰效率分析。此外，用MTT法检测细胞表面修饰对肿瘤细胞活力及其生长情况的影响。结果　GFP-SA重组融合蛋白在大肠杆菌实现了高效表达（约占细菌总蛋白的20%），通过纯化和复性制备的GFP-SA双功能融合蛋白具有双重活性，即：链亲和素介导的、对生物素高效特异的结合活性，和GFP发射绿色荧光的活性，并能高效修饰表面已生物素化的肿瘤细胞。此外，GFP-SA双功能融合蛋白的细胞表面修饰对细胞的活力及其生长无显著影响。结论　GFP-SA融合蛋白能高效修饰表面已生物素化的肿瘤细胞，可用作肿瘤疫苗研究的示踪蛋白及实验对照体系。

Study on Expression,Purification of GFP-SA Recombine Protein and Anchoring Carcinoma Cells

Objective GFP (green fluorescence protein)-streptavidin (SA) bi-functional fusion protein was generated and characterized in order to demonstrate our novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins. Methods GFP-SA/pET24 construct was generated and expressed in BL21(DE3) host bacteria at the high level. The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography, and then refolded. After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis. The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining. Results The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins. The GFP-SA bi-functional fusion protein exhibited the bi-functionality, i.e., SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence. The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly. Conclusion The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin, thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.